Development of an in vitro human lung organoid system to study alveolar type 2 stem cell maintenance and dysfunction during disease

Abstract

The human adult distal lung parenchyma is maintained by alveolar type 2 (hAT2) cells that have the ability to self-renew and differentiate into alveolar type 1 (hAT1) cells, a critical cell type involved in gas-exchange. Cellular damage within the alveoli can lead to severe lung dysfunction, and major alveolar defects are characteristic of a number of incurable lung diseases, including Idiopathic pulmonary fibrosis (IPF). The appearance of aberrant cell types within disease-associated honeycomb regions of the distal lung are a hallmark of human IPF. However, the role of hAT2 cells in IPF development has not been fully explored, partially due to current in vitro human alveolar models failing to maintain hAT2 identity, limiting their study. Therefore, development of alternative platforms are essential for elucidation of hAT2 maintenance during alveolar homeostasis and their potential dysfunction during disease.

Utilising primary adult hAT2 cells isolated from human distal lung samples, I successfully developed and characterised a novel in vitro 3D organoid platform that allowed for long-term hAT2 maintenance in chemically-defined conditions. Cultured hAT2-derived organoids underwent clonal expansion, were functionally mature and exhibited maintenance of differentiation capacity. High Wnt signalling and FGF7 presence were found to be essential for primary hAT2 self-renewal, while reduction of Wnt signalling led to hAT1 differentiation. To understand hAT2 dysregulation during disease, hypoxia was used to mimic an aspect of the IPF lung environment. Culture of healthy hAT2 cells in hypoxic conditions resulted in aberrant differentiation of hAT2 cells to SOX2-expressing airway-like cells. These cells exhibited higher proliferative capacity than normal hAT2 cells, with inhibition of Notch signalling resulting in a reduction in both the number of proliferative cells and SOX2+ organoids, indicating that hypoxia-induced induction of SOX2 in hAT2 cells may function through Notch signalling. Similar cells were also identified in honeycomb regions of human IPF patient lungs, implicating the hAT2 cell population as a potential origin for the ‘bronchiolisation’ observed in distal IPF lungs.

My results indicate that functional adult-derived hAT2 cells can be maintained long-term as 3D organoids in chemically-defined conditions. This novel culture system can be utilised to better understand both normal physiological and dysfunctional hAT2 stem cell behaviour. In addition, my data suggest that aberrant activation of signalling pathways implicated in IPF, such as hypoxia and Notch signalling, may function in causing incorrect hAT2 cell differentiation to airway-like cells. This could ultimately lead to a loss of correct alveolar stem cell maintenance, and a decline in lung function, both of which are observed in progressive, chronic lung diseases such as IPF.